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Distinct seasonal patterns of bacterioplankton abundance and dominance of

phyla alpha-Proteobacteria and Cyanobacteria in Qinhuangdao coastal waters
off the Bohai Sea

Article  in  Frontiers in Microbiology · August 2017

DOI: 10.3389/fmicb.2017.01579

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 ORIGINAL RESEARCH
published: 18 August 2017
doi: 10.3389/fmicb.2017.01579

Distinct Seasonal Patterns of

Bacterioplankton Abundance and
Dominance of Phyla
α-Proteobacteria and Cyanobacteria
in Qinhuangdao Coastal Waters Off
the Bohai Sea
Yaodong He 1† , Biswarup Sen 1† , Shuangyan Zhou 1 , Ningdong Xie 1 , Yongfeng Zhang 2 ,
Jianle Zhang 2 and Guangyi Wang 1,3*
1
Center for Marine Environmental Ecology, School of Environmental Science and Engineering, Tianjin University, Tianjin,
China, 2 Qinhuangdao Marine Environmental Monitoring Central Station, State Oceanic Administration, Qinhuangdao, China,
3
Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, China

Edited by:
Hongyue Dang, Qinhuangdao coastal waters in northern China are heavily impacted by anthropogenic
Xiamen University, China
and natural activities, and we anticipate a direct influence of the impact on the
Reviewed by:
Mohd Ikram Ansari,
bacterioplankton abundance and diversity inhabiting the adjacent coastal areas. To
King Abdullah University of Science ascertain the anthropogenic influences, we first evaluated the seasonal abundance
and Technology, Saudi Arabia patterns and diversity of bacterioplankton in the coastal areas with varied levels
Jérôme Hamelin,
Institut National de la Recherche of natural and anthropogenic activities and then analyzed the environmental factors
Agronomique (INRA), France which influenced the abundance patterns. Results indicated distinct patterns in
*Correspondence: bacterioplankton abundance across the warm and cold seasons in all stations. Total
Guangyi Wang
gywang@tju.edu.cn
bacterial abundance in the stations ranged from 8.67 × 104 to 2.08 × 106 cells/mL
† These authors have contributed
and had significant (p < 0.01) positive correlation with total phosphorus (TP), which
equally to this work. indicated TP as the key monitoring parameter for anthropogenic impact on nutrients
cycling. Proteobacteria and Cyanobacteria were the most abundant phyla in the
Specialty section:
This article was submitted to
Qinhuangdao coastal waters. Redundancy analysis revealed significant (p < 0.01)
Aquatic Microbiology, influence of temperature, dissolved oxygen and chlorophyll a on the spatiotemporal
a section of the journal
abundance pattern of α-Proteobacteria and Cyanobacteria groups. Among the 19
Frontiers in Microbiology
identified bacterioplankton subgroups, α-Proteobacteria (phylum Proteobacteria) was
Received: 17 February 2017
Accepted: 03 August 2017 the dominant one followed by Family II (phylum Cyanobacteria), representing 19.1–
Published: 18 August 2017 55.2% and 2.3–54.2% of total sequences, respectively. An inverse relationship
Citation: (r = −0.82) was observed between the two dominant subgroups, α-Proteobacteria
He Y, Sen B, Zhou S, Xie N,
Zhang Y, Zhang J and Wang G
and Family II. A wide range of inverse Simpson index (10.2 to 105) revealed spatial
(2017) Distinct Seasonal Patterns heterogeneity of bacterioplankton diversity likely resulting from the varied anthropogenic
of Bacterioplankton Abundance
and natural influences. Overall, our results suggested that seasonal variations impose
and Dominance of Phyla
α-Proteobacteria and Cyanobacteria substantial influence on shaping bacterioplankton abundance patterns. In addition, the
in Qinhuangdao Coastal Waters Off predominance of only a few cosmopolitan species in the Qinhuangdao coastal wasters
the Bohai Sea.
Front. Microbiol. 8:1579.
was probably an indication of their competitive advantage over other bacterioplankton
doi: 10.3389/fmicb.2017.01579 groups in the degradation of anthropogenic inputs. The results provided an evidence of

Frontiers in Microbiology | www.frontiersin.org 1 August 2017 | Volume 8 | Article 1579

He et al. Bacterioplankton Abundance and Diversity Patterns

their ecological significance in coastal waters impacted by seasonal inputs of the natural
and anthropogenic matter. In conclusion, the findings anticipate future development of
effective indicators of coastal health monitoring and subsequent management strategies
to control the anthropogenic inputs in the Qinhuangdao coastal waters.
Keywords: anthropogenic impacts, environmental variables, phylogenetic diversity, bacterioplankton abundance,
seasonal variations, redundancy analysis

INTRODUCTION the taxonomic diversity and abundance. A high concentration

Marine and coastal environments are of major concern
nowadays on account of the rapid developments and growth
in anthropogenic activities. Most commonly the anthropogenic
activities include terrestrial pollution, aquaculture, urban
development, tourism, maritime transport, agricultural and
industrial activities, oil refineries, and mining, etc. (Islam and
Tanaka, 2004). The nutrient inputs from these activities are the
major drivers of eutrophication in most of the coastal areas,
ultimately affecting the marine food web components (Vaquer-
Sunyer et al., 2015). In addition, rivers and allochthonous
biological processes also enrich coastal water with dissolved
compounds (Hiriart-Baer et al., 2008; Barrera-Alba et al.,
2009; Vargas et al., 2011; Gustafsson et al., 2014; Lafon et al.,
2014). Thus, organic matter mineralization and nutrient
cycling are crucial for maintenance of coastal ecosystem
health. In this context, the assemblage and abundance of
bacterioplankton involved in the marine microbial loop
(Farooq and Francesca, 2007) are canonical indicators of
ecosystem status and the extent of anthropogenic impact on the
coastal waters (Zhang et al., 2014). The spatial and temporal
dynamics of bacterioplankton community is driven by multiple
environmental factors such as latitudinal gradient (Fuhrman
et al., 2008; Milici et al., 2016), temperature (Kim et al., 2016),
predation (Oguz et al., 2013; Karus et al., 2014), nutrients
(Kim et al., 2016), succession (Chen H. et al., 2016), seasons
(Gilbert et al., 2012), and timescales (Fuhrman et al., 2015).
Apart from natural environmental factors, anthropogenic
activities also shape the bacterioplankton assemblage and
abundance pattern (Zhang et al., 2009; Thiyagarajan et al.,
2010; Zhou et al., 2011; Sauret et al., 2012; Jeffries et al.,
2016; Meziti et al., 2016). For instance, the abundances of
the bacterioplankton groups that increased and decreased
in the impacted sites were significantly correlated with
nutrients enrichment (Fodelianakis et al., 2014). Also, there
is a direct evidence of specific metal (Cd)-induced patterns in
bacterioplankton communities in coastal systems (Wang et al.,
2015). In oligotrophic coastal water microcosm with nitrate
perturbation, the bacterioplankton community composition
was greatly influenced by nitrate loading mode, indicative of
nitrate loading impact on marine environments (Dong et al.,
2017). Thus, nutrients and toxic pollutants enrichment seem to
be the important drivers of the bacterioplankton composition
in sites receiving direct human impact (Aguilo-Ferretjans et al.,
2008).
A closer inspection of the bacterioplankton community
dynamics reveals many interesting relationships and patterns in
of Synechococcus and low concentration of Prochlorococcus has
been observed in sites impacted by human activities with inputs
from the land (Bouvy et al., 2012). In a microcosm study,
it was found that anthropogenic impacts do not necessarily
influence the abundance pattern of rare species (<0.1% of relative
abundance), and in fact, nutrients enrichment increased the
relative abundance of only abundant species (>1% of relative
abundance) Polaribacter, Tenacibaculum, and Rhodobacteraceae
(Baltar et al., 2015). By the use of 16S rRNA gene cloning
and sequencing, the bacterioplankton diversity of oligotrophic
marine environments is relatively well studied than coastal
environments (Zhou et al., 2011; Ortega-Retuerta et al., 2014;
Hartmann et al., 2016; Milici et al., 2016; Dong et al.,
2017). Bacterioplankton diversity and abundance patterns on
a regional-scale and also for local sites under anthropogenic
impact remains a hot topic and several reports are available
(Simonato et al., 2010; Niu et al., 2011; Peierls and Paerl,
2011; Zhou et al., 2011; Fodelianakis et al., 2014; Laas et al.,
2014; Almutairi, 2015; Baltar et al., 2015; Meziti et al., 2015;
Xiong et al., 2015; Jeffries et al., 2016). Overall, the available
reports on bacterioplankton community composition at regional-
scale or in local sites indicate diverse patterns and complex
relationships between species composition and environmental
factors. In addition, phylogenetic analysis and abundance
patterns of bacterioplankton assemblages provide significant
insights into the bacterioplankton dynamics in the impacted
coastal areas which further leads us to the development of
coastal environmental monitoring and management strategies
(Demarcq et al., 2012). This calls for more research to identify
the bacterioplankton species that show strong linkages with
the implicit environmental factors for developing effective
descriptors of the anthropogenic impacts.
Qinhuangdao is a famous port city, an important energy
export port, and coastal tourist spot on the northwest coast of
the Bohai Sea in North China with an area of approximately
7812 km2 and a population of 2.9 × 106 (Gu et al., 2016). The
Bohai Sea coastline experiences the strong influence of riverine
systems, aquaculture, municipal sewage, industrial wastewaters,
and agricultural runoffs (Zhu et al., 2014). Land-use surrounding
the Qinhuangdao coastal area shifted drastically from agriculture
to industrialization in the last 30 years (Xu et al., 2010). The
area has undergone development of several petrochemical, steel,
and other projects, besides land reclamation and construction
of embankments (Zhu et al., 2014). The resulting land-use
change has overall influenced the coastal seawater nutrients
cycling and associated bacterioplankton, phytoplankton, and
other biotic factors (Xu et al., 2010; Liu et al., 2011). Results from

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He et al.
other and our previous studies have shown a distinct pattern
of bacterioplankton abundance and diversity as a consequence
of environmental influence such as estuaries, mariculture, and
rainfall (Li J. et al., 2011; Li et al., 2013; Shang et al., 2016; Zhou
et al., 2016). These reports further encourage us to investigate
closely the relationship between the bacterioplankton community
and nutrient levels across different seasons in the near-shore
and off-shore stations near Qinhuangdao coast and address
the following questions. What are the patterns in abundance
of major taxonomic groups and their spatial diversity? What
drives the spatiotemporal variations of bacterioplankton in the
Qinhuangdao coastal waters?
The objectives of the present study were to: (1) analyze the
abundance of total bacteria and major bacterioplankton groups
across different seasons, (2) identify the key environmental
factors that influence the bacterioplankton abundance, and (3)
assess the bacterioplankton distribution and diversity along the
near-shore and off-shore coastal areas of Qinhuangdao.
MATERIALS AND METHODS
Study Area and Water Collection
Qinhuangdao, one of the highly urbanized regions, is a port
city on the coast of China located in the northeastern Hebei
province, and it is situated about 280 km east of Beijing on
the northwest Coast of Bohai Sea, the innermost gulf of Yellow
Sea. The Qinhuangdao coastal area has a humid continental
climate with four distinct seasons. It has a monsoon-influenced
humid continental climate with the highest precipitation
(152–189 mm) during July–August period. From October month
the temperature starts to fall (<5◦ C) until April, and a very
low (<10 mm) precipitation occurs during November to March
period (Gu et al., 2016).
Six stations (W1–W6) were selected that had the distinct
influence of the local environment (Figure 1). The W1 station
is located outside the port of Qinhuangdao mainly polluted by
the shipping industry and municipal sewage, the W3 station
is located in the Xinkai river estuary near the Beidaihe Forest
Wetland and Qinhuangdao Wildlife Park, and the W5 station
is located between the Yang River Estuary and Dai River
Estuary. The Yang River and Dai River are severely polluted by
microorganisms and the total number of bacteria exceeds the
standard levels. W3 and W5 stations are also located in the tourist
area and near the bathing beach. W2, W4, and W6 are off-
shore stations with aquaculture facility near W6 station. Water
samples were collected in the month of March, July, October,
and December (2014) at the stations W1, W2, W3, W4, W5,
W6 (Figure 1). For each month one water sample (1 L) at 1 m
depth from the surface was collected using a sample hydrophore.
A plexiglass hydrophore (JC-800, Juchuang, China) of capacity
5 L, diameter 15 cm, and height 37.5 cm was used as the sample
hydrophore. All samples were stored in sterile glass bottles at
4◦ C and then transported to the laboratory. A 500 mL volume
of water sample was filtered using 0.22 µm membrane filter
(AmeriTech Inc., United States). The membrane filters coated
with microorganisms were stored at −80◦ C.
Frontiers in Microbiology | www.frontiersin.org
Bacterioplankton Abundance and Diversity Patterns
Water Quality and Nutrient Analysis
The pH, temperature, dissolved oxygen (DO), and salinity of the
water samples at six stations were measured using a portable YSI
Pro Plus Multiparameter instrument (YSI Inc., United States).
The contents of total nitrogen (TN), total phosphorus (TP),
ammonia nitrogen (NH4 -N), nitrate nitrogen (NO3 -N), nitrite
nitrogen (NO2 -N) and Chlorophyll a (Chl a) were determined
in water samples in accordance with the “Specification for
oceanographic survey” (GB/T 12763.4-2007). Briefly, NH4 -N
was measured by sodium bromate oxidation method which
involves oxidization of ammonium salt to nitrite in alkaline
medium, followed by determination of the TN content using
diazo spectrophotometry. After deducting the concentration of
NO2 -N, the concentration of NH4 -N was estimated. NO3 -N
was measured by zinc cadmium reduction method where nitrate
is quantitatively reduced to nitrite in water by zinc cadmium
reduction method following determination of total nitrite by the
diazo and azo method, the original nitrite was then corrected
and the nitrate content was calculated. NO2 -N was measured
by a diazo azo method which involves the reaction of nitrite
with sulfanilamide in acidic medium following product reaction
with hydrochloric acid and synthesis of a red azo dye whose
absorbance was measured at 543 nm. Chl a measurement
was done by an extraction-fluorimetric method which involves
excitation by blue light of the acetone extract of Chl a to produce
red fluorescence (Holm-Hansen and Bo, 1978). The fluorescence
value of the extract was determined at 685 nm before and after
acidification.
The abundance of total bacteria at the 6 stations near
Qinhuangdao coastal area over warm and cold seasons were
monitored using fluorescence microscopy to assess the influence
of anthropogenic and natural activities in the near-shore
and off-shore waters. The total bacterial count in water
samples (formaldehyde-fixed) at each station was estimated by
fluorescence microscopy (Eclipse Ni-U, Nikon Instruments Inc.,
United States). Sample (2 mL water) filtration was done at a
pressure of 150 mm Hg through a polycarbonate filter (0.22 µm
aperture, 25 mm diameter) (AmeriTech Inc., United States).
The membrane filter was stained with 2X SYBR-Gold solution
(300 µl) and was visualized at 480–485 nm wavelength (blue light
excitation). Data reported are the means of counts in 20 randomly
selected fields per sample. For each station, triplicate samples
were processed and for each replicate sample, bacterial numbers
were counted in 20 randomly selected fields. The data reported
are the means of counts in triplicate samples.
DNA Isolation and Sequence Analysis of
Bacterioplankton Assemblage
Five hundred milliliter of surface water samples collected in July
2014 from 6 stations were filtered onto 0.2 µm polycarbonate
membrane filters (AmeriTech Inc., United States). The resulting
filters were stored in ultra-low temperature refrigerator (−80◦ C)
until DNA isolation. The total DNA was extracted using
E.Z.N.A.TM Water DNA Kit (Omega Bio-Tek Inc., United States)
following the manufacturer’s instructions. The isolated DNA was
used as template for bacterial 16S rRNA gene amplification using
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He et al. Bacterioplankton Abundance and Diversity Patterns

FIGURE 1 | Map of sampling stations.

universal primers 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) 72◦ C 1 min; 35 cycles; 72◦ C 10 min. PCR amplification for
and 1492R (50 -GGTTACCTTGTTACGACTT-30 ) on Bio-Rad each sample was performed in triplicates and the triplicate PCR
S1000 thermal cycler (Bio-Rad Laboratories Inc., United States) products were mixed to obtain the final sample for clone library
using the following protocol: 95◦ C 5 min; 94◦ C 45 s, 55◦ C 45 s, construction. All PCR products were gel-purified, cloned into

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He et al.
a pMD18-T Simple Vector (Sino Biological Inc., China), and
then transformed into Escherichia coli DH5α competent cells by
traditional heat shock treatment. 16S rRNA clone libraries of a
total of six water samples (one sample per station) for the month
of July were constructed and 90 positive clones were randomly
picked from each sample plate for sequence analysis (Beijing
Genomics Institution, China). The coverage of the clone libraries
ranged from 31.1 to 56.7% and the impact of low coverage on
diversity analysis was minimized by calculating Simpson diversity
estimate (Haegeman et al., 2013) in Mothur software package
Plasmid Midi
(Schloss et al., 2009). The sequences have been submitted to
NCBI Genebank database with accession number MF498066 –
MF498485.
All sequence analyses were performed within versions 1.35.1
of the Mothur software package (Schloss et al., 2009). The
FASTA format sequences were aligned with SILVA alignments
and the distance was calculated at cutoff 0.03 (97% sequence
homology) using align.seqs and dist.seqs commands, respectively.
Chimeras were identified using chimera.uchime command with
default parameters. Sequences were classified using classify.seqs
command with reference to Ribosomal Database Project
(RDP) as the taxonomy file (silva.bacteria.rdp.tax) and the
remove.lineage command was used to remove the sequences
that belong to mitochondria, chloroplast, eukaryote, archaea,
and unknown lineages. Cluster command was used to assign
sequences to OTUs with opticlust clustering method. α-diversity
was calculated by counting the number of OTUs and using
the reciprocal of Simpson Index (invsimpson). The invsimpson
calculator is preferred to other measures of α-diversity as it
indicates the richness in a community with a uniform evenness
that would have the same level of diversity. Moreover, the inverse
of the Simpson index has some biological interpretation and
do not tend to be affected by sampling effort because it is
independent of abundance distributions (Haegeman et al., 2013).
Quantitative PCR Analysis of
Bacterioplankton Groups
To assess the phylum/class level abundance distribution of total
bacteria in warm and cold seasons, qPCR experiments with
phylum and class specific primers (Supplementary Table S1) were
conducted for all 6 stations. qPCR method was used to detect
and quantitate the abundance of the major phylogenetic groups
of bacterioplankton in the water samples from Qinhuangdao
coastal area. The advantage of qPCR method in our study
was its ability to quantitate specific bacterioplankton groups
by using phylum/class specific primers. In addition, qPCR
quantitation is fast, reliable, and accurate. Based on the results
of clone library analysis and identification, the following
dominant classes were selected to carry out quantitative PCR
experiments: α-Proteobacteria, β-Proteobacteria, Cyanobacteria,
Actinobacteria, Firmicutes, and Bacteroidetes. PCR was carried
out in a 20 µL reaction mixture containing 1 µL DNA template
(2.8-10.5 ng/µL), 10 µL SYBR Select Master Mix (Thermo Fisher
Scientific Inc.), 1 µL forward primer (10 nmol/mL), 1 µL reverse
primer (10 nmol/mL), and 7 µL nuclease-free molecular-grade
water. The qPCR cycling condition was: 95◦ C 55 s; 95◦ C 20 s,
Frontiers in Microbiology | www.frontiersin.org
Bacterioplankton Abundance and Diversity Patterns
60◦ C 35 s, 72◦ C 30 s; 40 cycles; 95◦ C 1 min, 60◦ C 1 min,
heating-up from 75 to 95◦ C, the increment of 0.5◦ C for 8 s. The
annealing temperature of each primer is shown in Supplementary
Table S1. The standard curves were constructed with known
amounts of plasmid DNA containing the sequence insert. For
the positive plasmid preparation, the PCR products were ligated
to pMD18-T Vector by pMD18-T Vector Cloning Kit (Takara
Biotechnology (Dalian) Co., Ltd.) and then transformed into
E. coli competent cells (DH5α) followed by a screening of positive
clones. Plasmid DNA was extracted using E.Z.N.A
R
Kit (Omega Bio-Tek Inc., United States) and the concentration
of the extracted DNA was measured using NanoDrop ND-1000
spectrophotometer (Nanodrop Technology). The gene copies
were calculated using the mean mass of pMD18-T Vector ligated
with the target DNA sequence. DNA of each sample and standard
plasmid DNA dilution (101 and 105 ) were used as templates for
qPCR. Each sample and standard were analyzed in triplicates.
The standard curve was constructed to estimate the copy number
of each sample according to the dilution ratio and copy number
of the standard plasmid template.
Statistical Analysis
Principal component analysis (PCA) was done to reveal
the relationships between the environmental parameters and
the distribution of the sampling stations in an ordination
plot. Pearson’s correlation analysis was performed to assess
the correlation between the abundance of total bacteria,
major bacterioplankton groups, and environmental parameters.
Redundancy analysis (RDA) was carried out to identify
the explanatory environmental parameters that significantly
(p < 0.01) constrain the bacterioplankton abundance data by
forward selection in Canoco version 5.02. RDA was performed
using abundance (qPCR data) and environmental data. The
PCA of environmental parameters was done using normalized
(z-score) data in Canoco version 5.02. For RDA, the qPCR data
were log(x+1) transformed and the environmental data was
normalized by z-score.
RESULTS
High Seasonal Variations of
Environmental Parameters
The environmental parameters across 6 stations near
Qinhuangdao coastal area appeared highly heterogeneous
spatiotemporally. In the warm season (July–October), the
temperature of coastal waters in all stations was within 21–25◦ C,
whereas in the cold season (December–March) the temperature
was significantly lower ranging from −0.3 to 6.8◦ C. pH was
notably higher (11.6 ± 0.09) in October at all stations. Salinity
levels were similar (∼29 ppt) in all stations and also during
warm and cold seasons. The DO levels were relatively higher
in cold period than warm as a result of temperature effect on
oxygen solubility in water. The average values of environmental
parameters (nutrients) with their variation over different periods
at the 6 stations near Qinhuangdao coastal area are shown
in Table 1. The NH4 -N level was high in W5 station than
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He et al.
TABLE 1 | The data of environmental parameters at near-shore (W1, W3, and W5) and off-shore (W2, W4, and W6) stations near Qinhuangdao coastal area.
Stations NH4 -N (mg/L) NO2 -N ( mg/L)
W1 0.067 (0.037) 0.035 (0.024)
W3 0.061 (0.032) 0.024 (0.017)
W5 0.104 (0.048) 0.028 (0.018)
W2 0.053 (0.012) 0.024 (0.017)
W4 0.041 (0.015) 0.024 (0.022)
W6 0.054 (0.017) 0.024 (0.019)
TN, TP, and Chl a indicate total nitrogen, total phosphate, and Chlorophyll a.
Mean (SD) of values over 4 seasons (July, October, December, and March) are provided.
that of the others. In October the NH4 -N was relatively high
at all stations (0.05–0.17 mg/L). No significant variation in
NO2 -N and NO3 -N content was observed at the 6 stations.
TN was detected in all stations (0.35–0.6 mg/L) with relatively
large fluctuations (0.6–1.8mg/L) in the warm season (July)
and was lowest in the cold season (December). In July and
March, the TP was found to be comparatively higher than in
October and December. Furthermore, the average TP content
was higher in W1, W2, W3, W5 (0.12–0.13 mg/L) than that in
W4 and W6 stations (0.07–0.08 mg/L). W1 and W5 stations
showed highest average Chl a level (11.6–16.0 mg/m3 ) followed
by W3 and W6 (5.6–5.9 mg/m3 ) and lowest levels at W2
and W4 (2.6-3.2 mg/m3 ). The variation in Chl a level was
substantially less in W2 and W4 stations suggesting a low
abundance of phytoplankton at these stations than at the
polluted stations (W1, W3, W5, and W6). In the Bohai Sea,
silicates and Si/dissolved inorganic N ratio have been associated
with phytoplankton abundance (Chen Y.H. et al., 2016). In
addition, climate and mariculture activity have a significant
correlation with the Chl a trends (Fu et al., 2016). River discharge
and suspended sediment also influence Chl a in Bohai Sea coast.
These factors might have played a role in the spatial variation
of Chl a observed in the present study. Also, Chl a mostly was
higher in July in all stations probably due to more growth of
phytoplankton resulting from optimal light and temperature
conditions.
By analyzing all the measured environmental variables in
combination, the resultant ordination plot showed distinct
partitioning of coastal water samples across warm and cold
seasons rather than across near-shore and off-shore stations
(Figure 2). The ordination plot could explain 59.6% of total
variation in the environmental data and revealed a linear
positive correlation between TN, temperature, and Chl a,
and a negative correlation of DO with these variables. These
parameters were most crucial in the partitioning of samples
from the warm and cold seasons. Similarly, NH4 -N and NO3 -
N had a positive correlation with each other, and salinity,
NO2 -N, and TP did not show any notable correlations to
any of the other variables. Overall, the PCA biplot clearly
showed that seasonal variation of the measured environmental
parameters was more than spatial variation. Thus, seasonal
loads of anthropogenic inputs seem to have a significant
impact on the nutrients conditions in the coastal areas of
Qinhuangdao.
Frontiers in Microbiology | www.frontiersin.org
Bacterioplankton Abundance and Diversity Patterns
NO3 -N (mg/L) TN (mg/L) TP (mg/L) Chl a (mg/m3 )
0.337 (0.196) 0.793 (0.199) 0.131 (0.080) 11.604 (6.334)
0.355 (0.419) 1.103 (0.542) 0.126 (0.077) 5.603 (5.389)
0.337 (0.248) 0.959 (0.447) 0.128 (0.019) 16.004 (10.559)
0.435 (0.431) 1.158 (0.506) 0.121 (0.079) 3.209 (1.181)
0.362 (0.296) 0.851 (0.334) 0.079 (0.054) 2.651 (1.826)
0.331 (0.189) 0.937 (0.461) 0.069 (0.063) 5.873 (8.444)
Bacterioplankton Abundance Patterns
and Influencing Factors
The results of fluorescence microscopy based detection of total
bacterial abundance in the near-shore and off-shore stations
are shown in Figure 3. In July, all the stations mostly showed
highest abundance (5.32 × 105 – 1.71 × 106 cells/mL) of bacteria
compared to other months. Overall, the total bacterial abundance
in each of the 6 stations over the 4 months was in between
8.67 × 104 and 2.08 × 106 cells/mL. Within the other 3 months,
March recorded higher abundance than October and December
in all the stations. The stations W1, W3, W5, and W6 typically
had relatively more abundance than the W2 and W4 perhaps due
to nutrients overload in these stations as a consequence of higher
anthropogenic and natural activities as evident from their higher
level of Chl a. A significant (p < 0.01) positive correlation of total
bacterial abundance with TP was seen (Figure 4).
The qPCR based abundance estimates of 6 major phylum/class
i.e., α-Proteobacteria, β-Proteobacteria, Cyanobacteria,
Actinobacteria, Firmicutes, and Bacteroidetes are shown in
Figure 5. α-Proteobacteria, Actinobacteria, Firmicutes, and
Bacteroidetes showed a fairly similar pattern of abundance
distribution in the 6 stations. Notably, in the warm season, these
4 groups exhibited relatively higher abundances than in cold
season. However, β-Proteobacteria and Cyanobacteria showed
distinct trends, wherein the former one showed a similar pattern
in all stations over the 4 months, i.e., higher abundance in cold
than warm season. Bacteroidetes abundance at W1 and W6
stations remained high in October, and in W5 its abundance
increased only in July and December. Near-shore and off-shore
differences in the abundance of the major phylotypes were also
apparent. In the warm season, near-shore stations possessed
more Cyanobacteria and Bacteroidetes than off-shore stations.
Alternatively, in cold months, near-shore stations possessed
a relatively higher abundance of α-Proteobacteria, Firmicutes,
Actinobacteria, and β-Proteobacteria than off-shore stations.
Table 2 shows the Pearson’s correlation coefficients for the
relationship between bacterioplankton abundance and some
environmental parameters. α-Proteobacteria showed significant
(p < 0.05) positive correlation with TN, Chl a, DO, and
temperature. Furthermore, from the ordination plot (Figure 6)
based on redundancy analysis, temperature and Chl a were the
major factors that positively correlated with the first principal
component and were the significant explanatory parameters
(p < 0.01) that constrained the abundance of α-Proteobacteria
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He et al. Bacterioplankton Abundance and Diversity Patterns

FIGURE 2 | Biplot of the principal component analysis (PCA) for environmental parameters in the sampling stations near Qinhuangdao coastal area. The two
principal components (PC1 and PC2) explained 59.64% of total variation in environmental data, and shows clear partitioning of July and October samples from
December and March along the PC1, and partitioning of July from October along PC2. PC1 and PC2 strongly correlated to variables NH4 -N and NO3 -N, and total
nitrogen (TN), temperature (T) and Chlorophyll-a (Chl a), respectively. Total phosphate (TP), NO2 -N, and salinity were not correlated to PC1 and PC2. Blue arrows
represent environmental parameters, and circles in color represents sampling points.

and Cyanobacteria phylotypes. On the contrary, DO showed a
negative influence (p < 0.01) and salinity was non-significant in
explaining any variation of the abundance of bacterioplankton
groups. Overall, samples collected in warm July or October had
a much higher abundance of α-Proteobacteria, Firmicutes, and
Actinobacteria than that of cold March or December. In contrast,
β-Proteobacteria, Cyanobacteria, and Bacteroidetes did not show
any distinct seasonal influence which likely suggests their diverse
metabolic potential and ability to utilize organic matter even at
low concentrations that allow them to attain invariable growth
throughout the year.
Spatial Distribution and Diversity of
Bacterioplankton Assemblages
The assemblage of bacterioplankton communities featuring
diversity and distribution at the 6 stations in the warm season
was assessed and the results are reported in Table 3 and Figure 7.
Based on the environmental data, July exhibited greater levels
and fluctuations in the measured parameters perhaps due to
more nutrient inputs that resulted from anthropogenic and
natural activities in warm season. Thus, further characterization
of bacterioplankton phylogeny, composition, and diversity
was conducted on water samples collected in July. Out of
the selected clones analyzed, 62, 62, 68, 44, 47, and 71 OTUs
were identified in W1, W2, W3, W4, W5, and W6 stations,
respectively (Table 3). A broad phylogenetic distribution of
the bacterioplankton community at the 6 stations in warm
season is shown in Figure 7A. The analysis of 16S rRNA
gene sequences from these stations revealed 7 distinct phyla
(Figure 7A). The majority of the sequences generated from
the 6 bacterioplankton clone libraries were affiliated to phyla
Proteobacteria (21.7–75.9%), Cyanobacteria (4.6–54.2%),
Bacteroidetes (9–21.2%), Actinobacteria (1.1–5.6%), Firmicutes

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He et al.
FIGURE 3 | Fluorescence microscopy based quantification of total bacteria at
the near-shore and off-shore stations near the Qinhuangdao coastal area of
Bohai Sea. ‘W1’ to ‘W6’ indicate the different sampling stations.
FIGURE 4 | Significant (p < 0.01) correlation between total bacterial
abundance and total phosphorous. Values on Y -axis are shown in log-scale.
(0–4.4%), Verrucomicrobia (0–3.5%), Planctomycetes (0–3.3%).
The unclassified group represented sequences in the range
of 9.2–26.7%. At the phylum level Proteobacteria (49% of
total sequences), Cyanobacteria (24% of total sequences), and
Bacteroidetes (17% of total sequences) were the dominant groups
in all the stations. There were 19 bacterioplankton classes in the
total sequences acquired from the coastal waters of Qinhuangdao
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Bacterioplankton Abundance and Diversity Patterns
(Figure 7B). The predominant phylum Proteobacteria comprised
of five classes namely α-Proteobacteria, γ-Proteobacteria,
ε-Proteobacteria, β-Proteobacteria, and δ-Proteobacteria
accounting to ca. 82, 9, 3.6, 2, and 1.6% of Proteobacteria,
respectively. Further taxonomic resolution of the assemblages
revealed nearly 30 different families that were assigned to
the identified OTUs from the bacterioplankton assemblages
(Supplementary Figure S1). The OTUs within Proteobacteria
phylum were assigned to 15 families: Campylobacteraceae,
Comamonadaceae, Methylophilaceae, Pseudobacteriovoracaceae,
Burkholderiaceae, Bacteriovoracaceae, Moraxellaceae,
Halomonadaceae, Oceanospirillaceae, Enterobacteriaceae,
Rhodospirillaceae, SAR11, Hyphomicrobiaceae, Rhodobiaceae,
and Rhodobacteraceae. The second major phylum Cyanobacteria
comprised OTUs which were all assigned to a single family —
Family II. The OTUs representing phylum Bacteroidetes belonged
to 4 families: Flavobacteriaceae, Cryomorphaceae, Saprospiraceae,
and Chitinophagaceae. Rhodobacteraceae represented major
proportion (>10% of total 16S r RNA sequences) in all the
stations followed by Family II especially in W1 and W4 stations.
Only three OTUs assigned to Rhodobacteraceae, Family II, and
Cryomorphaceae were cosmopolitans, i.e., they were found in all
the stations. Overall, α-Proteobacteria (Rhodobacteraceae) (19.1–
55.2%) and Cyanobacteria (Family II) (2.3–54.2%) were the
most dominant cosmopolitan groups. Interestingly, it was noted
that these two dominant groups had an inverse relationship
(r = −0.82).
Based on inverse Simpson index (1/D), the ascending order of
diversity at the 6 stations was W5 < W4 < W1 < W6 < W2 < W3
ranging from 10.2 to 105 (Table 3). The results suggested a wide
difference in the bacterioplankton diversity, which likely resulted
from the presumed shifts in the environmental conditions of the
coastal waters of Qinhuangdao. The inverse Simpson index (1/D)
may serve as a sensitive indicator of the anthropogenic impact in
the Bohai Sea and can accurately monitor the extent of human
activities in Qinhuangdao coastal region.
DISCUSSION
Spatiotemporal Variations and Factors
Influencing Bacterioplankton Abundance
Most global-scale studies on the biogeography of
bacterioplankton communities have revealed intriguing
findings and have largely shown that diversity and abundance
pattern are closely linked to latitudinal gradient (Fuhrman
et al., 2008), seasons (Cuevas et al., 2004; Fuhrman et al., 2015),
temperature and nutrient levels (Pommier et al., 2007; Vichi
et al., 2007). In addition, previous studies have well established
that bacterioplankton abundance is potentially linked to elevated
anthropogenic nutrient loads in aquatic ecosystems. Overall,
nutrient contents (TP, TN, Chl a, and DO) and gradient of
the natural parameter (temperature) displayed significant
differences across seasons and sampling stations in our study.
In the warm season, we observed a greater concentration of
nutrients (TN, TP, and Chl a) and decreased DO which is
consistent with other studies (Lee et al., 2011; Shang et al.,
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He et al. Bacterioplankton Abundance and Diversity Patterns

FIGURE 5 | 16S rRNA gene abundance (qPCR quantification) of major phylogenetic groups from coastal bacterioplankton assemblages in 6 stations near
Qinhuangdao coastal area. The symbols and lines in black, red, green, blue, cyan, and magenta represent stations W1, W2, W3, W4, W5, and W6. Values on Y -axis
are plotted in log-scale.

2016). The heavy rainfall in the warm season seems one of
the main causes which overload organic matter and pollutants
through the estuaries and run-offs. Freshwater inflows and
aquaculture activities also deliver nutrients and contaminants
besides nutrients from fertilizers and anthropogenic activities,
which may also cause the nutrient concentration variations, and
shape the bacterioplankton abundance pattern (Jeffries et al.,
2016). Generally, low salinity in coastal water indicates that the
area has received river discharge, however, in the present study
any significant lowering of salinity in the coastal waters was
not observed. This perhaps is an indication that Qinhuangdao
coastal area does not experience the heavy impact of riverine
system round the year. In addition to nutrient inputs, the
direct input of pathogenic and fastidious microorganisms from
wastewater treatment, wildlife sanctuary, and sewage sources,
which are known to influence the bacterioplankton abundance

TABLE 2 | Pearson’s correlation analysis showing correlations between abundance of total bacteria, bacterioplankton groups and some environmental parameters.

Bacterial groups Total nitrogen (mg/L) Total phosphate (mg/L) Chlorophyll-a (mg/m3 ) Dissolved oxygen (mg/L) Temperature (◦ C)

Total bacteria NS 0.719∗∗ (0.00) NS NS NS

α-Proteobacteria 0.490∗ (0.015) NS 0.461∗ (0.023) NS 0.974∗∗ (0.00)
β-Proteobacteria NS NS 0.532∗∗ (0.007) NS NS
Cyanobacteria NS NS NS NS NS
Actinobacteria NS NS NS NS NS
Bacteroidetes NS NS NS NS 0.410∗ (0.046)
Firmicutes NS NS NS NS NS
∗∗ P < 0.01, ∗P< 0.05; NS, non-significant.
The values represents correlation coefficient (significant bilateral) for sample number N = 24. Total bacteria and bacterioplankton group abundance data were based on
fluorescence microscopy and quantitative PCR, respectively.

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He et al. Bacterioplankton Abundance and Diversity Patterns

FIGURE 6 | Biplot of redundancy analysis (RDA) showing relationship between the environmental variables and 16S rRNA gene abundance (qPCR quantification) of
major phylogenetic groups in six stations near Qinhuangdao coastal area. The two RDA axes (RDA1 and RDA2) explained 70.35% of total variation in abundance
data, and the significant (p < 0.01) explanatory variables were temperature (temp), dissolved oxygen (DO), and Chlorophyll a (Chl_a). Blue arrows represent different
phylotypes, gray arrows represent environmental parameters, stars represent near-shore samples and circles represent off-shore samples. Different colors of the star
or circle symbols represent different sampling time, with March samples in green, July samples in red, October samples in yellow and December samples in purple.

(Vandewalle et al., 2012; Liu et al., 2015) also might affect strong correlation in total phosphorous and abundance
bacterioplankton abundance and diversity in Qinhuangdao agree well with previous findings that describe inorganic
coastal waters. phosphorous as an essential nutrient and that it serves
Our results demonstrated that bacterioplankton abundance a vital role in cellular energy storage and transformation
in the Qinhuangdao coastal waters exhibit substantial shifts and is also a growth-limiting nutrient (Brandsma, 2016).
over time and relatively less in space. Furthermore, the Although bacterioplankton and phytoplankton abundance
(Chl a) in many studies are closely linked, our study show no
relationship between them, an uncoupling phenomenon often
TABLE 3 | The number of 16S rRNA sequences, operational taxonomic units observed when inorganic phosphorus is available (Robarts and
(OTUs), and inverse Simpson diversity (1/D) estimates for the bacterioplankton
Carr, 2009). The considerable reduction in bacterioplankton
clone libraries.
abundance in October perhaps is an indication of anthropogenic
Stations 16S rRNA Operational Inverse Simpson inputs or short-term physical forcing events that bring in
Sequence taxonomic diversity index nutrients which offer phytoplankton an opportunity to outgrow
number units (OTUs) (1/D)
bacterioplankton. The higher abundance of bacterioplankton
W1 87 62 34.9 in warm July and cold March is probably associated with
W2 85 62 83.0 dissolved organic carbon of algal exudates from algal bloom and
W3 80 68 105.0 allochthonous carbon, respectively. Overall, this study extends
W4 82 44 16.3 upon the mechanism of bacterioplankton dynamics and explains
W5 85 47 10.2 the consistency of abundance pattern with the utilization
W6 89 71 70.3 of specific substrates. Nevertheless, the tight connection of
W1–W6 represents the 6 stations near Qinhuangdao coastal area for which abundance with TP bespeaks high implication in coastal
bacterioplankton clone libraries were constructed and analyzed. pollution monitoring.

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He et al.
FIGURE 7 | Composition and distribution of bacterioplankton assemblages at the 6 stations near Qinhuangdao coastal area. Percentage distribution of the 16S
rRNA gene sequences at the (A) phylum level and (B) class level indicates higher relative abundance of subgroups Proteobacteria, Cyanobacteria, Bacteroidetes,
and Actinobacteria.
Bacterioplankton abundance in the near-shore area is larger
than that in the open ocean, especially in the area of the
Gulf and the estuarine area. In our study, generally the near-
shore abundance was higher than off-shore, perhaps caused
by the uneven distribution of organic matter in the coastal
stations that are affected directly by run-offs, sewage, human
activities, etc. than off-shore stations. As the seawater gets
polluted by industrial wastewater and/or domestic sewage,
the bacterioplankton may go in a state of unstable nutrition
which subsequently affects the total bacterioplankton abundance,
transiently. In our study, the abundance was relatively higher
in the stations adjacent to river estuaries than that of other
stations, while within the near-shore stations, the difference was
not significant. Interestingly, in station W5 the bacterioplankton
abundance fluctuated dramatically that possibly indicated a
strong influence of nearby river estuaries heavily polluted
with invasive microorganisms. The results showed that the
total abundance in the stations near Qinhuangdao coastal area
fluctuated between 8.67 × 104 (W1 in March) to 2.08 × 106 (W4
in October) cells/ml which is in good agreement with the estimate
projected by Whitman et al. (Whitman et al., 1998) and empirical
data (Al-Rifaie et al., 2008; Robarts and Carr, 2009; Li H.-Y. et al.,
2011).
Although total abundance pattern provides an indication
of anthropogenic impacts on coastal waters to a certain
extent, knowing the pattern of variation in the composition
of bacterioplankton allows further understanding of how the
growth of different groups of bacterioplankton is controlled
by sporadic nutrient inputs. In addition, different phylogenetic
groups of bacterioplankton play a specialized role in their
consumption of nutrients, and thus dissecting their abundance
fluctuations would provide greater insight into the extent of
nutrient loading and their recycling by functional groups. The
distinct pattern of seasonal variations in the abundance of
α-Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes
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Bacterioplankton Abundance and Diversity Patterns
groups most likely explains their role in organic matter
transformation as chemoheterotrophs. These groups exhibited
higher abundance mostly in warm season when there were
intense anthropogenic activities and subsequent enrichment of
seawater with pollutants. In fact, it was shown that warming
promotes bacterioplankton to benefit from the phytoplankton
bloom and increase their abundance ultimately shifting toward
a more heterotrophic system (Scheibner et al., 2014). These
groups perhaps are directly involved in the decomposition of
anthropogenic chemicals and algal exudates via a microbial
loop as evident from their strong relationship with TN
and Chl a. In contrast, β-Proteobacteria was found to be
tightly coupled with Chl a, which probably indicated that
this less known bacterioplankton group feeds mostly on the
algal exudates and may not be directly involved in the
decomposition of complex polymers. Bacteroidetes also take part
in organic matter degradation and have been associated with
the occurrence of algal bloom. Some members of this group use
proteorhodopsin to capture light energy for growth making them
photoheterotrophic (Gomez-Consarnau et al., 2007). The strong
correlation between Bacteroidetes group and temperature could
likely be a direct upshot of more light intensity in the warm
season.
In our study, temperature, TN, Chl a, and DO were
identified as key drivers of spatiotemporal dissimilarity between
bacterioplankton abundance patterns in the coastal waters of
Qinhuangdao area. Salinity, temperature, and pH are naturally
varying parameters, while nutrient concentrations and DO
levels indicate the extent of anthropogenic activities besides
allochthonous inputs. Temperature and salinity are strong drivers
of variations in microbial assemblages of estuaries (Jeffries et al.,
2016), but in coastal water salinity changes are marginal, and
therefore non-significant in explaining community variations.
Previous studies have reported strong association of TN, Chl a,
temperature, and TP with the microbial community composition
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He et al.
variations and perhaps are the major drivers of community
dynamics in aquatic ecosystems (Fodelianakis et al., 2014;
Wang et al., 2015; Jeffries et al., 2016; Dong et al., 2017).
However, in the present study, these key parameters were
tenuous in partitioning the near-shore and off-shore waters near
Qinhuangdao area, perhaps indicating the influence of other
factors such as Si/dissolved inorganic N ratio, river discharge,
short-term physical forcing events, and suspended sediments.
In conclusion, this study shows temporal variations in the
environmental parameters were more pronounced than spatial in
shaping the abundance patterns of bacterioplankton inhabiting
the coastal waters of Qinhuangdao. This further connotes the
consequence of sporadic anthropogenic activities and their
chemical inputs in influencing the bacterioplankton assemblages
in the harbor area. Our study provided the first insight into
the bacterioplankton ecology of an anthropogenically impacted
regional-scale coastal water of Qinhuangdao, by combining
bacterioplankton abundance patterns with physicochemical
characteristics.
Spatial Patterns of Bacterioplankton
Composition and Diversity
The regional and global ocean sampling expeditions have
highlighted the importance of bacterioplankton community
dynamics and their assembly into functional communities
by elucidating their community structure inhabiting different
marine locations (Yutin et al., 2007; Lauro et al., 2014).
Yet, the information on bacterioplankton diversity within
littoral areas adjoining harbors is quite limited perhaps due to
complex interactions of multitudinous factors that underline
their structure and distribution. As far as the littoral area of Bohai
Sea is concerned, this is one of the first studies to our knowledge
which reports the diversity of the bacterioplankton assemblage
nearby harbor and the factors that shape their structure
and composition. Proteobacteria, Cyanobacteria, Actinobacteria,
and Bacteroidetes represented the major phylotypes at the
6 stations, and is in agreement with other reports which
have found these bacterioplankton groups to be the major
inhabitant of marine habitats (Li J. et al., 2011; Li et al., 2013;
Smedile et al., 2014; Xiong et al., 2015; Jeffries et al., 2016).
Proteobacteria and Cyanobacteria exhibited higher dynamics in
their composition among others. Although it is quite challenging
to relate phylogenetic diversity and ecological function in marine
microbial diversity, yet it is hypothesized that these major groups
efficiently metabolize exudates and dissolved organic matter
composed of carbohydrates and/or amino acids derived from
both growing and senescent phytoplankton (Teira et al., 2008). In
addition, these groups are also known to degrade hydrocarbons
and inorganic compounds from allochthonous sources (Robarts
and Carr, 2009).
Within phylum Proteobacteria, the class α-Proteobacteria
represented the majority of the 16S rRNA gene sequences and
was the predominant group in all stations. α-proteobacteria
are usually abundant in coastal waters and they can form
the predominant surface- and particle-colonizing group (Dang
and Lovell, 2002a,b). Rhodobacteraceae (also called the marine
Roseobacter clade) is usually the largest subgroup in the
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Bacterioplankton Abundance and Diversity Patterns
α-proteobacteria and they are the pioneering and dominant
microorganisms on submerged surfaces and organic particles
(Dang and Lovell, 2000; Dang and Lovell, 2016). The class
γ-Proteobacteria constituted only a minor fraction in our
molecular-based study, although members of this class are
best known for their rapid growth and also represented as a
major group in global surveys (Pommier et al., 2007). OTUs
assigned to α-Proteobacteria and Cyanobacteria phyla were
cosmopolitans in our study, and this finding is consistent
with previous reports (Pommier et al., 2007; Smedile et al.,
2014). Cyanobacteria dominated at W1 and W4 stations and
most OTUs belonged to the genus Synechococcus. They are
mainly free-living and are the main participants and contributors
to primary productivity of the global carbon cycle and are
the common bacteria inhabiting coastal, temperate, and cold
environments (Brown et al., 2014; Xia et al., 2015). From
genome analysis, Synechococcus sp. WH8102 was found to
be able to use some new organic compounds as sources of
nitrogen and phosphorus (Palenik et al., 2003). In addition,
Cyanobacteria (Synechococcus) are typically more abundant in
higher nutrients or dynamic conditions and are often described
as generalist better suited to exploit the fluctuating environments
(Palenik et al., 2003). In contrast, Rhodobacteraceae comprise
mainly aerobic photo- and chemo-heterotrophs besides purple
non-sulfur bacteria which perform photosynthesis in anaerobic
environments. Members of this subgroup are metabolically
heterogeneous and are capable of anoxygenic phototrophy,
aromatic and organosulfur degradation (Lenk et al., 2012).
They are the main groups involved in the demethylation
of dimethylsulfoniopropionate in the water column (Curson
et al., 2011). They are largely involved in sulfur and carbon
biogeochemical cycling and symbiosis with other micro- and
macro-organisms (Pujalte et al., 2014). The dominance of
Rhodobacteraceae in the coastal waters of Qinhuangdao port area
epitomizes the degradation of organic and inorganic compounds
containing sulfur. The inverse relationship between the two
most predominant taxonomic groups (Rhodobacteraceae and
Family II) may be either due to competition, niche partitioning,
or another mechanism. Further investigation is needed to
ascertain the exact cause of the inverse relationship. The low
proportion of γ- and δ-Proteobacteria at the 6 stations perhaps
indicate the absence of chronic polyaromatic hydrocarbons
(PAHs) influence in the coastal waters near Qinhuangdao
port, because these groups are often seen associated with
high levels of chronic PAHs (Jeanbille et al., 2016). Moreover,
the SAR11 considered as highly diverse and abundant at
different depths and across habitat types within euphotic and
mesopelagic zones was detected only in few stations (W1,
W2, and W5). The absence of SAR11 clade at other stations
perhaps indicates their rarity and thus this clade was unable
to represent in our clone libraries. Nevertheless, the use of
inverse Simpson Index which does not tend to be affected
by rare species could predict diversity more accurately than
other estimators of diversity, and thus diversity analysis in
our study was relatively robust and could give a reliable and
meaningful interpretation. The wide range and high variation
in diversity estimate (1/D) indicate environmental heterogeneity
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He et al.
of the Qinhuangdao coastal waters due to varied nutrient types
and their concentrations that directly influence the relative
abundance of bacterioplankton functional groups.
In the present study, the richness (species number) of the
bacterioplankton community was not estimated taking into
account the high rate of estimation error with low coverage
libraries (Haegeman et al., 2013). Instead, we implemented the
estimation of Simpson diversity index which is a robust estimator
and provides an accurate estimation of the species diversity. It
is independent of any species abundance distributions (SADs)
unlike the richness estimators (Haegeman et al., 2013). The use
of Simpson index in our study assured accurate estimation of
bacterioplankton diversity even with low coverage clone libraries.
Also, as explained in an earlier study, coverage does not correlate
with library size and the authors clearly mentioned that “a large
library size did not guarantee a stable estimate of phylotype
richness, nor was a small library necessarily too small” (Kemp
and Aller, 2004). The authors also showed that the average size
of 220 bacterial libraries was 81 ± 10, ranging in size from 5 to
417 clones. Therefore, our study with 90 clones per library is in
good agreement with the average size 81 ± 10 (Kemp and Aller,
2004).
Overall, our study on the bacterioplankton diversity at
stations near Qinhuangdao port suggests prominence of only
two cosmopolitan OTUs with several endemic OTUs. The
predominance of few species is probably an indication of
selective advantage of these species to overcome a load of
chemical inputs despite scatting grazing and predation by virus
and macro-organisms. Also, the effect of anthropogenic and
allochthonous inputs was evident from the wide range of inverse
Simpson diversity estimates for bacterioplankton assemblages at
the stations near Qinhuangdao port. This is consistent with the
notion that disturbed marine and coastal environments possess
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AUTHOR CONTRIBUTIONS
YH has contributed to data acquisition, analysis and drafting the
work. BS has contributed to data interpretation, analysis, drafting
the work and critical revision. SZ, YZ, and JZ have contributed
to data acquisition. NX helped in data analysis. GW contributed
to conception of the work, data interpretation and manuscript
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and accountable for all aspects of the work.
ACKNOWLEDGMENTS
The work was partially supported by NSFC (grant # 31670044 and
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of the funding agencies or any of its subagencies.
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